Method for improving pharmacokinetics

ABSTRACT

The object of the present invention is to inhibit oxidative metabolism of a compound of formula I by co-administration with ritonavir, a cytochrome P450 inhibitor.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/766,051 filedApr. 23, 2010 which claims the benefit of priority to U.S. ProvisionalApplication 61/172,722 filed Apr. 25, 2009 the contents of both areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an improved method or administering acompound according to formula I for treating HCV by co-administrationwith ritonavir. The invention further relates to a pharmaceuticalcomposition containing the compound of formula I and ritonavir.

BACKGROUND

Hepatitis C virus (HCV) is the leading cause of chronic liver diseasethroughout the world. (Boyer, N. et al., J. Hepatol. 2000 32:98-112).The World Health Organization (WHO) estimates that more than 170 millionpeople worldwide (or about 3% of the world's population) are infectedwith the single-stranded ribonucleic acid (RNA) HCV. (G. M. Lauer and B.D. Walker, N. Engl. J. Med. 2001 345:41-52) Approximately one-fifth ofchronically infected patients with HCV will eventually develop cirrhosisof the liver, suffering considerable morbidity and mortality, includingliver failure and hepatocellular carcinoma (T. J. Liang et al. Ann.Intern. Med. 2000 132:296-305; M. W. Fried et al. N. Engl. J. Med. 2002347:975-982). HCV infection is the primary indication for livertransplantation in the United States (NIH Consensus Statement onManagement of Hepatitis C. 2002 June 10-12 19(3):146;http://www.ncbi.nlm.nih.gov/pubmed/14768714).

HCV has been classified as a member of the virus family Flaviviridaethat includes the genera flaviviruses, pestiviruses, and hapaceiviruseswhich includes hepatitis C viruses (Rice, C. M., Flaviviridae: Theviruses and their replication. In: Fields Virology, Editors: B. N.Fields, D. M. Knipe and P. M. Howley, Lippincott-Raven Publishers,Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped viruscontaining a positive-sense single-stranded RNA genome of approximately9.4 kb. The viral genome consists of a highly conserved 5′ untranslatedregion (UTR), a long open reading frame encoding a polyprotein precursorof-approximately 3011 amino acids, and a short 3′ UTR.

Genetic analysis of HCV has identified six main genotypes which divergeby over 30% of the DNA sequence. More than 30 subtypes have beendistinguished. In the US approximately 70% of infected individuals haveType 1a and 1b infection. Type 1b is the most prevalent subtype in Asia.(X. Forns and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J. Bukhet al., Semin. Liv. Dis. 1995 15:41-63). Unfortunately type 1 infectionis more resistant to therapy than either type 2 or 3 genotypes (N. N.Zein, Clin. Microbiol. Rev., 2000 13:223-235).

The HCV genome encodes a polyprotein of 3010-3033, amino acids (Q. L.Choo, et al., Proc. Natl. Acad. Sci. USA, 1991 88:2451-2455; N. Kato etal., Proc. Natl. Acad. Sci. USA 1990 87:9524-9528; A. Takamizawa et al.,J. Virol. 1991 65:1105-1113). Viral structural proteins include anucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2.HCV also encodes two proteases, a zinc-dependent metalloproteinaseencoded by the NS2-NS3 region and a serine protease encoded in the NS3region. The HCV NS3 protease is a serine protease that helps process themajority of the viral enzymes, and is thus considered essential forviral replication and infectivity. These proteases are required forcleavage of specific regions of the precursor polyprotein into maturepeptides. The carboxyl half of nonstructural protein 5, NS5B, containsthe RNA-dependent RNA polymerase.

Currently a limited number of approved therapies are available for thetreatment of HCV infection. New and existing therapeutic approaches fortreating HCV infection and inhibiting of HCV NS5B polymerase activityhave been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; DiBesceglie, A. M. and Bacon, B. R., Scientific American, October: 199980-85; G. Lake-Bakaar, Curr. Drug Targ. Infect. Dis. 2003 3(3):247-253;P. Hoffmann et al, Exp. Opin. Ther. Patents 2003 13(11):1707-1723; M. P.Walker et al., Exp. Opin. Investing. Drugs 2003 12(8):1269-1280; S.-L.Tan et al., Nature Rev. Drug Discov. 2002 1:867-881; J. Z. Wu and Z.Hong, Curr. Drug Targ-Infect. Dis. 2003 3(3):207-219.

Ribavirin(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]triazole-3-carboxylicacid amide; Virazole®) is a synthetic, non-interferon-inducing,broad-spectrum antiviral nucleoside analog. Ribavirin has in vitroactivity against several DNA and RNA viruses including Flaviviridae(Gary L. Davis. Gastroenterology 2000 118:S104-S114). Although, inmonotherapy ribavirin reduces serum amino transferase levels to normalin 40% of patients, it does not lower serum levels of HCV-RNA. Ribavirinalso exhibits significant toxicity and is known to induce anemia.Viramidine is a ribavirin prodrug converted ribavirin by adenosinedeaminase to in hepatocytes. (J. Z. Wu, Antivir. Chem. Chemother. 200617(1):33-9)

Interferons (IFNs) have been available for the treatment of chronichepatitis for nearly a decade. IFNs are glycoproteins produced by immunecells in response to viral infection. Two distinct types of interferonare recognized: Type 1 includes several interferon alphas and oneinterferon beta, type 2 includes interferon gamma. Type 1 interferonsare produced mainly by infected cells and protect neighboring cells fromde novo infection. IFNs inhibit viral replication of many viruses,including HCV, and when used as the sole treatment for hepatitis Cinfection, IFN suppresses serum HCV-RNA to undetectable levels.Additionally, IFN normalizes serum amino transferase levels.Unfortunately, the effects of IFN are temporary. Cessation of therapyresults in a 70% relapse rate and only 10-15% exhibit a sustainedvirological response with normal serum alanine transferase levels.(Davis, Luke-Bakaar, supra)

Combination therapy of HCV with ribavirin and interferon-α □ currentlyis the standard of care for treatment-naïve patients HCV. Combiningribavirin and PEG-IFN (infra) results in a sustained viral response(SVR) defined as undetectable hepatitis C virus ribonucleic acid (HCVRNA) 24 weeks after completion of therapy (M. W. Fried et al. supra) in54-56% of patients with type 1 HCV. The SVR approaches 80% for type 2and 3 HCV. (Walker, supra) Furthermore, PEG-IFN is given by injection,and the hematologic and constitutional toxicities of PEG-IFN and of RBVare difficult for many patients to tolerate for the long (up to 48weeks) duration of treatment required. Currently, there is no SOCtreatment for patients who either relapsed or did not respond to(nonresponders) PEG-IFN/RBV therapy. Given the high prevalence of CHCdisease worldwide, the high treatment failure rate with the current SOC,and tolerability issues with the current SOC, there is a substantialunmet medical need to improve and expand therapeutic options for thesepatient populations. The effectiveness of the host defenses is hamperedby the ability of HCV to disrupt, evade, and antagonize the host immuneresponse, not only ensuring continued viral infection, but also quiteoften resisting the antiviral action of IFN therapy (M. Gale, Jr. and E.M. Foy, Nature 2005. 436:939-945). Therefore, a strategy that targetsthe virus itself may improve the results of therapy in comparison withcurrent therapy options.

A number of potential new molecular targets for drug development asanti-HCV therapeutics have now been identified including, but notlimited to, the N52-N53 autoprotease, the NS3 protease, the NS3 helicaseand the NS5B polymerase. The RNA-dependent RNA polymerase is absolutelyessential for replication of the single-stranded, positive sense, RNAgenome. This enzyme has elicited significant interest among medicinalchemists.

HCV polymerase inhibitors are another target for drug discovery andcompounds in development include R-1626, R-7128, IDX184/IDX102,PF-868554 (Pfizer), VCH-759 (ViroChem), GS-9190 (Gilead), A-837093 andA-848837 (Abbot), MK-3281 (Merck), GSK949614 and GSK625433 (Glaxo),ANA598 (Anadys), VBY 708 (ViroBay).

Inhibitors of the HCV NS3 protease also have been identified aspotentially useful for treatment of HCV. Protease inhibitors in clinicaltrials include VX-950 (Telaprevir, Vertex), SCHSO3034 (Broceprevir,Schering), TMC435350 (Tibotec/Medivir) and ITMN-191 (Intermune). Otherprotease inhibitors in earlier stages of development include MK7009(Merck), BMS-790052 (Bristol Myers Squibb), VBY-376 (Virobay),IDXSCA/IDXSCB (Idenix), BI12202 (Boehringer), VX-500 (Vertex), PHX1766Phenomix).

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a method forincreasing the bioavailability and or blood level of a Hepatitis C virusNS3 protease inhibitor according to formula I in a patient comprisingco-administering to the patient the compound of formula I and acytochrome P450 monooxygenase inhibitor.

In another aspect of the present invention there is provided apharmaceutical composition comprising a compound of formula I and acytochrome P450 monooxygenase inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

As used in this specification, whether in a transitional phrase or inthe body of the claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of a compoundor composition, the term “comprising” means that the compound orcomposition includes at least the recited features or components, butmay also include additional features or components.

The term “optional” or “optionally” as used herein means that asubsequently described event or circumstance may, but need not, occur,and that the description includes instances where the event orcircumstance occurs and instances in which it does not.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valueof the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value of the numerical range, including theend-points of the range. As an example, a variable which is described ashaving values between 0 and 2, can be 0, 1 or 2 for variables which areinherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other realvalue for variables which are inherently continuous.

There is a need for compositions and therapeutic combinations fortreating HCV. Combination chemotherapy has proven effective in treatmentof HCV, however, patient compliance improves as the number of doses andfrequency of administration decreases. Improved compounds and dosageregimens would be useful in anti-HCV therapies. The subject inventionprovides a method for administering a compound of formula I (R7227) anda CYP 3A4 inhibitor that enhances the bioavailability of R7227 andreduces the amount of R7227nthat must be administered.

Compound I (R7227) is a highly potent and selective macrocyclic,peptidomimetic inhibitor of NS3/4A protease activity selected fordevelopment as an oral agent for the treatment of HCV infection due tothe potency displayed in inhibiting NS3/4A protease. R7227 displayssignificant exposure in the liver of animal species, which is theprimary or sole site of HCV replication in humans, and has an acceptabletoxicologic profile. R7227 is a highly potent inhibitor of NS3/4Aproteolysis with a 50% inhibitory concentration (IC₅₀)≦0.225 nM and ahigh degree of specificity for the intended target. In cell-basedpotency assays employing a genotype 1 HCV replicon, R7227 has an IC₅₀ of1.77 nM. R7227 additionally displays synergistic antiviral effects withpolyethylene glycol conjugated (“pegylated”) interferon alfa-2a(PEG-IFN-α2a, Pegasys®, Roche) in this same cell-based assay thatsuggests R7227 will be useful in HCV therapy.

Cytochromes, especially the CYP3A4 isoform, have been found tometabolize R7227 resulting in a requirement for more frequent and higherdose levels to maintain therapeutically effective blood levels.

In early preclinical studies, cytochrome P450 phenotyping using chemicalinhibitors suggests that multiple CYP isozymes including 3A4, 2C19, 1A2,2D6, and 2C9 participate in the metabolism of (R7227). Furtherexperiments with recombinant CYPs show that only CYP3A4 metabolizedR7227 to an extent that could influence the pharmacokinetics. Therefore,a cytochrome P450 monooxygenase inhibitor in an amount effective toinhibit metabolism of the protease inhibitor could increase thebioavailability of R7227 compared to administration in the absence ofthe CYP inhibitor.

Some drugs are metabolized by cytochrome P450 enzymes. These enzymestypically oxidize the drugs resulting in unfavorable pharmacokineticcharacteristics (e.g., decreased blood levels, decreased half-life). Inthese cases inhibition of drug metabolism can lead to improvements inthe pharmacokinetic profile of the drug. (see, e.g., U.S. Pat. No.6,037,157; D. E. Kempf et al. Antimicrob. Agents Chemother., 199741:654-660; W. J. Curatolo and G. Foulds, U.S. Patent Publication2004/0091527 and M. G. Cordingley, U.S. Patent PublicationUS2004/0152625). To ascertain whether combination therapy with acytochrome P450 antagonist will improve the pharmacokinetics of a drugthe metabolic pathways involved must be elucidated.

Cytochrome P450 (CYP P450) is a very large and diverse superfamily ofhemoproteins. Both exogenous and endogenous compounds as substrates forcytochrome P450 isoforms. Cytochrome P450 3A4 (CYP3A4; EC 1.14.13.97),is one of the most important enzymes involved in the metabolism ofxenobiotics in the body. CYP3A4 is involved in the oxidation of thelargest range of substrates of all the CYPs. Although CYP3A4 ispredominantly found in the liver, it is also present in other organs andtissues of the body.

Any CYP inhibitor that improves the pharmacokinetics of the relevant NS3protease may be used in a method of this invention. These CYP inhibitorsinclude, but are not limited to, ritonavir (WO 94/14436), ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole,cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine,fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir,fosamprenavir, saquinavir, lopinavir, delavirdine and erythromycin. Apreferred CYP inhibitor is ritonavir.

Ritonavir is a potent inhibitor of CYP3A4 activity and is currentlyutilized at low non-therapeutic doses (e.g., 100 mg twice daily) toenhance or “boost” the PK of other HIV protease inhibitors (PIs). Giventhe high prevalence of HIV/HCV co-infection, R7227 could be used for thetreatment of HCV in HIV/HCV co-infected patients who receivedritonavir-boosted HIV PIs, resulting in possible interactions betweenritonavir-boosted HIV PIs and R7227.

However, in addition to the inhibitory effect on 3A4, ritonavir appearsto induce the activities of other enzymes including CYPs 1A2, 2C9,and2C19. Although during acute dosing, the inhibitory effect ofritonavir will be predominant resulting in elevated levels of R7227,chronic dosing anticipated in HCV therapy could induce other isoformswhich could oxidize R7227 offsetting the desired inhibitory effect.

In one embodiment of the present invention there is provided a methodfor increasing the bioavailability of Hepatitis C virus NS3/4A proteaseinhibitor R7227 in a patient in need thereof comprising administeringR7227 and a cytochrome P450 monooxygenase inhibitor wherein the amountof the cytochrome P450 monooxygenase inhibitor is sufficient to elevatethe blood levels of R7227 compared to the blood levels R7227 in theabsence of a cytochrome P450 monooxygenase inhibitor.

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising administering R7227 and ritonavir. Ritonavir is marketed byAbbott Laboratories under the name of NORVIR® as an HIV proteaseinhibitor (Chemical Abstract Registry Number 1555213-67-5).

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising co-administering R7227 and ritonavir wherein R7227 andritonavir are in a separate dosage form. The doses of each can be takeneither at or about the same time or the doses may be taken at differentintervals.

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising co-administering R7227 and ritonavir wherein R7227 andritonavir are administered simultaneously. R7227 and ritonavir may bepresent in a single formulation for increased patient convenience.

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising co-administering R7227 and ritonavir wherein R7227 andritonavir are administered in a single dosage form.

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising co-administering a dose of 25 to 600 mg/day of R7227 and 50to 400 mg/day of ritonavir.

In another embodiment of the present invention there is provided amethod for increasing the bioavailability of R7227 in a patientcomprising co-administering a dose of 50 to 300 mg/day of R7227 and 100to 200 mg/day of ritonavir.

In a another embodiment of the present invention there is provided amethod for treating HCV comprising administering to a patient in needthereof the compound of formula I, or free base or otherpharmaceutically acceptable salt thereof, and a cytochrome P450monooxygenase inhibitor.

In a another embodiment of the present invention there is provided amethod for treating HCV comprising administering to a patient in needthereof the compound of formula I, or free base or otherpharmaceutically acceptable salt thereof, and ritonavir.

Combination therapy has proven to be a valuable component of antiviraltherapy and therefore treatment of HCV with R7227 and ritonavir maycomprise administration of another component comprising an additionalagent selected from an immunomodulatory agent; an antiviral agent;another HCV protease inhibitor; an inhibitor of another target in theHCV life cycle; such as an HCV polymerase inhibitor or combinationsthereof

In another embodiment of the present invention there is provided amethod for treating HCV comprising co-administering along with acompound of formula I and ritonavir at least one additional agentselected from an immunomodulatory agent and/or an antiviral agent and/oranother inhibitor of HCV NS3/4A protease and/or an inhibitor of NS5Bpolymerase and/or a broad-spectrum viral inhibitor and/or anothercytochrome P-450 inhibitor.

In another embodiment of the present invention there is provided amethod for treating HCV which method comprises co-administering II(R7128) along with R7227 and ritonavir.

In still another embodiment of the present invention there is provided amethod for treating HCV which method comprises co-administering α-, β-or γ-interferon and/or thymosin and/or ribavirin and/or R7128 along withR7227 and ritonavir.

In a another embodiment of the present invention there is provided apharmaceutical composition comprising R7227 or a pharmaceuticallyacceptable salt thereof, a cytochrome P450 monooxygenase inhibitor or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient, diluent or carrier.

In a another embodiment of the present invention there is provided apharmaceutical composition comprising R7227, or free base or otherpharmaceutically acceptable salt thereof, a cytochrome P450monooxygenase inhibitor, and at least one pharmaceutically acceptableexcipient, diluent or carrier.

In yet another embodiment of the present invention there is provided acomprising R7227, or free base or other pharmaceutically acceptable saltthereof, a cytochrome P450 monooxygenase inhibitor, the HCV polymeraseinhibitor R7128 and at least one pharmaceutically acceptable excipient,diluent or carrier.

In a another embodiment of the present invention there is provided apharmaceutical composition comprising R7227, or free base or otherpharmaceutically acceptable salt thereof, ritonavir, or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient, diluent or carrier.

In a fifteenth embodiment of the present invention there is provided akit comprising a Hepatitis C virus NS3 protease inhibitor according toformula I and ritonavir.

In an embodiment of the present invention there is provided apharmaceutical pack containing comprising a compound according toformula I, ritonavir, and optionally an informational insert containingdirections for the use of the inhibitors.

Ritonavir low dose was reported to increase the exposure of midazolam,the most sensitive CYP3A probe substrate, by approximately 7-fold. (A.A. Mathias et al., Clin. Pharmacol. Ther. 2009 85(1)64-70). SimCYP®simulation (SymCYP Limited, Blades Enterprise Centre, John Street,Sheffield S2 4SU, UK) predicted that ritonavir could increase R7227exposure between ca. 2- and 4-fold, assuming the contribution of CYP3Ato the overall elimination of R7227 is 50% and 100%, respectively. Whenco-administered with ritonavir in this study, the predicted 4-foldincrease in R7227 exposure is still significantly lower than thatobserved at the highest safe and tolerable dose of 1600 mg in the SADstudy in healthy volunteers.

Multiple doses of ritonavir 100 mg every 12 hours significantlyincreased R7227 AUC_(0→∞), C_(max), and C_(12 h) by approximately5.5-fold, 3.25-fold, and 27- to 42-fold, respectively. The multiple-doseeffect of ritonavir on R7227 C₁₂h is less than that of the acutesingle-dose effect of ritonavir possibly due to the induction of CYPenzymes by ritonavir following multiple dosing offsetting some of theacute inhibitory effect by ritonavir on CYP 3A4. Thus thepharmacokinetics R7227 was substantially improved by theco-administration of ritonavir.

Dosage and Administration

The compounds of the present invention may be formulated in a widevariety of oral administration dosage forms and carriers. Oraladministration can be in the form of tablets, coated tablets, dragées,hard and soft gelatin capsules, solutions, emulsions, syrups, orsuspensions. Compounds of the present invention are efficacious whenadministered by other routes of administration including continuous(intravenous drip) topical parenteral, intramuscular, intravenous,subcutaneous and suppository administration, among other routes ofadministration. The preferred manner of administration is generally oralusing a convenient daily dosing regimen which can be adjusted accordingto the degree of affliction and the patient's response to the activeingredient.

A compound or compounds of the present invention, as well as theirpharmaceutically useable salts, together with one or more conventionalexcipients, carriers, or diluents, may be placed into the form ofpharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds or principles, and the unit dosage forms may containany suitable effective amount of the active ingredient commensurate withthe intended daily dosage range to be employed. The pharmaceuticalcompositions may be employed as solids, such as tablets or filledcapsules, semisolids, powders, sustained release formulations, orliquids such as solutions, suspensions, emulsions, elixirs, or filledcapsules for oral use; or in the form of suppositories for rectal orvaginal administration; or in the form of sterile injectable solutionsfor parenteral use. A typical preparation will contain from about 5% toabout 95% active compound or compounds (w/w). The term “preparation” or“dosage form”is intended to include both solid and liquid formulationsof the active compound and one skilled in the art will appreciate thatan active ingredient can exist in different preparations depending onthe target organ or tissue and on the desired dose and pharmacokineticparameters.

The term “excipient” as used herein refers to a compound that is usefulin preparing a pharmaceutical composition, generally safe, non-toxic andneither biologically nor otherwise undesirable, and includes excipientsthat are acceptable for veterinary use as well as human pharmaceuticaluse. The compounds of this invention can be administered alone but willgenerally be administered in admixture with one or more suitablepharmaceutical excipients, diluents or carriers selected with regard tothe intended route of administration and standard pharmaceuticalpractice.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for human pharmaceutical use.

A “pharmaceutically acceptable salt” form of an active ingredient mayalso initially confer a desirable pharmacokinetic property on the activeingredient which were absent in the non-salt form, and may evenpositively affect the pharmacodynamics of the active ingredient withrespect to its therapeutic activity in the body. The phrase“pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier may beone or more substances which may also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier generally is a finely divided solid which is a mixture withthe finely divided active component. In tablets, the active componentgenerally is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired. Suitable carriers include but are not limited to magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Solid form preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Liquid formulations also are suitable for oral administration includeliquid formulation including emulsions, syrups, elixirs, aqueoussolutions, aqueous suspensions. These include solid form preparationswhich are intended to be converted to liquid form preparations shortlybefore use. Emulsions may be prepared in solutions, for example, inaqueous propylene glycol solutions or may contain emulsifying agentssuch as lecithin, sorbitan monooleate, or acacia. Aqueous solutions canbe prepared by dissolving the active component in water and addingsuitable colorants, flavors, stabilizing, and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell known suspending agents.

The compounds of the present invention may be formulated for parenteraladministration (e.g., by injection, for example bolus injection orcontinuous infusion) and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, forexample solutions in aqueous polyethylene glycol. Examples of oily ornonaqueous carriers, diluents, solvents or vehicles include propyleneglycol, polyethylene glycol, vegetable oils (e.g., olive oil), andinjectable organic esters (e.g., ethyl oleate), and may containformulatory agents such as preserving, wetting, emulsifying orsuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilisation from solution for constitutionbefore use with a suitable vehicle, e.g., sterile, pyrogen-free water.

The compounds of the present invention may be formulated foradministration as suppositories. A low melting wax, such as a mixture offatty acid glycerides or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The compounds of the present invention may be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient. For example, the compounds of the present invention can beformulated in transdermal or subcutaneous drug delivery devices. Thesedelivery systems are advantageous when sustained release of the compoundis necessary and when patient compliance with a treatment regimen iscrucial. Compounds in transdermal delivery systems are frequentlyattached to an skin-adhesive solid support. The compound of interest canalso be combined with a penetration enhancer, e.g., Azone(1-dodecylaza-cycloheptan-2-one). Sustained release delivery systems areinserted subcutaneously into to the subdermal layer by surgery orinjection. The subdermal implants encapsulate the compound in a lipidsoluble membrane, e.g., silicone rubber, or a biodegradable polymer,e.g., polyactic acid.

Suitable formulations along with pharmaceutical carriers, diluents andexpcipients are described in Remington: The Science and Practice ofPharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19thedition, Easton, Pa. A skilled formulation scientist may modify theformulations within the teachings of the specification to providenumerous formulations for a particular route of administration withoutrendering the compositions of the present invention unstable orcompromising their therapeutic activity.

The modification of the present compounds to render them more soluble inwater or other vehicle, for example, may be easily accomplished by minormodifications (salt formulation, esterification, etc.), which are wellwithin the ordinary skill in the art. It is also well within theordinary skill of the art to modify the route of administration anddosage regimen of a particular compound in order to manage thepharmacokinetics of the present compounds for maximum beneficial effectin patients.

The methods described herein comprise administration of combinations ofa Hepatitis C virus NS3/4A protease inhibitor and a cytochrome P450monooxygenase inhibitor. Such administration is referred to herein asco-administration. Co-administration includes administering eachinhibitor in the same dosage form or in different dosage forms. Whenadministered in different dosage forms, the inhibitors may beadministered at the same or at different times and in any order.Accordingly, this invention provides methods wherein the CYP inhibitoris administered together with the Hepatitis C virus NS3/4A proteaseinhibitor in the same dosage form or in separate dosage forms.

If the CYP inhibitor and protease inhibitor are administered in separatedosage forms, each inhibitor may be administered about simultaneously.Alternatively, the CYP inhibitor may be administered in any time periodaround administration of the protease inhibitor. That is, the CYPinhibitor may be administered prior to, together with, or following theNS3/4A protease inhibitor. The time period of administration should besuch that the CYP inhibitor affects the metabolism of the proteaseinhibitor. For example, if the protease inhibitor is administered first,the CYP inhibitor should be administered before the protease inhibitoris metabolized and/or excreted

The term “therapeutically effective amount” as used herein means anamount required to reduce symptoms of the disease in an individual. Thelevel therapeutic of effectiveness in HCV therapy is generallydetermined by measuring the levels of viral RNA. The dose will beadjusted to the individual requirements in each particular case. Thatdosage can vary within wide limits depending upon numerous factors suchas the severity of the disease to be treated, the age and general healthcondition of the patient, other medicaments with which the patient isbeing treated, the route and form of administration and the preferencesand experience of the medical practitioner involved.

Dosage levels of between about 100 and about 800 mg per day, preferablybetween about 200 and about 600 mg per day of the NS3 protease inhibitorare useful for the prevention and treatment of HCV mediated disease. Forthe CYP inhibitor, the dosage levels of between about 50 to about 400 mgper day, would be typical. More typical would be dosage levels ofbetween about 100 to about 200 mg per day. Typically, the pharmaceuticalcompositions of, and according to, this invention will be administeredfrom about 1 to about 2 times per day. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect for the individual patient is reached. One of ordinaryskill in treating diseases described herein will be able, without undueexperimentation and in reliance on personal knowledge, experience andthe disclosures of this application, to ascertain a therapeuticallyeffective amount of the compounds of the present invention for a givendisease and patient.

For preferred dosage forms of ritonavir have been disclosed by L. A.Al-Razzak et al. in U.S. Pat. No. 5,484,801 published Jan. 16, 1996,U.S. Pat. No. 5,948,436 published Sep. 7, 1999, WO 95/07696 publishedMar. 23, 1995 and WO 95/09614 published Apr. 13, 1995.

The methods described herein comprise administration of combinations ofa Hepatitis C virus NS3 protease inhibitor and a cytochrome P450monooxygenase inhibitor. Such administration is referred to herein asco-administration. Co-administration includes administering eachinhibitor in the same dosage form or in different dosage forms. Whenadministered in different dosage forms, the inhibitors may beadministered at the same or at different times and in any order.Accordingly, this invention provides methods wherein the CYP inhibitoris administered together with the Hepatitis C virus NS3/4A proteaseinhibitor in the same dosage form or in separate dosage forms.

If the CYP inhibitor and protease inhibitor are administered in separatedosage forms, each inhibitor may be administered about simultaneously.Alternatively, the CYP inhibitor may be administered in any time periodaround administration of the protease inhibitor. That is, the CYPinhibitor may be administered prior to, together with, or following theNS3/4A protease inhibitor. The time period of administration should besuch that the CYP inhibitor affects the metabolism of the proteaseinhibitor. For example, if the protease inhibitor is administered first,the CYP inhibitor should be administered before the protease inhibitoris metabolized and/or excreted

Combination therapy has proven to be a valuable component of antiviraltherapy and therefore treatment of HCV with R7227 and ritonavir maycomprise administration of another component comprising an additionalagent selected from an immunomodulatory agent; an antiviral agent;another HCV protease inhibitor; an inhibitor of HCV polymerase oranother target in the HCV life cycle or combinations thereof.

Pharmaceutical compositions may also be prescribed to the patient in“patient packs” containing the whole course of treatment in a singlepackage, usually a blister pack. Patient packs have an advantage overtraditional prescriptions, where a pharmacists divides a patient'ssupply of a pharmaceutical from a bulk supply, in that the patientalways has access to the package insert contained in the patient pack,normally missing in traditional prescriptions. The inclusion of apackage insert has been shown to improve patient compliance with thephysician's instructions.

Administration of the combination of the invention by means of a singlepatient pack, or patient packs of each formulation, containing within apackage insert instructing the patient to the correct use of theinvention is a desirable additional feature of this invention which canimprove patient compliance.

According to a further aspect of the invention is a pack comprising atleast a NS3 protease inhibitor and a CYP 3A4 inhibitor of the inventionand an information insert containing directions on the use of thecombination of the invention. In an alternative embodiment of thisinvention, the pharmaceutical pack further comprises one or more ofadditional agent as described herein. The additional agent or agents maybe provided in the same pack or in separate packs.

Another aspect of this involves a packaged kit for a patient to use inthe treatment of HCV infection or in the prevention of HCV infection,comprising: a single or a plurality of pharmaceutical formulation ofeach pharmaceutical component; a container housing the pharmaceuticalformulation(s) during storage and prior to administration; andinstructions for carrying out drug administration in a manner effectiveto treat or prevent HCV infection.

Accordingly, this invention provides kits for the simultaneous orsequential administration of a NS3/4A protease inhibitor and a CYPinhibitor (and optionally an additional agent) or derivatives thereofare prepared in a conventional manner. Typically, such a kit willcomprise, e.g. a composition of each inhibitor and optionally theadditional agent(s) in a pharmaceutically acceptable carrier (and in oneor in a plurality of pharmaceutical formulations) and writteninstructions for the simultaneous or sequential administration.

In another embodiment, a packaged kit is provided that contains one ormore dosage forms for self administration; a container means, preferablysealed, for housing the dosage forms during storage and prior to use;and instructions for a patient to carry out drug administration. Theinstructions will typically be written instructions on a package insert,a label, and/or on other components of the kit, and the dosage form orforms are as described herein. Each dosage form may be individuallyhoused, as in a sheet of a metal foil-plastic laminate with each dosageform isolated from the others in individual cells or bubbles, or thedosage forms may be housed in a single container, as in a plasticbottle. The present kits will also typically include means for packagingthe individual kit components, i.e., the dosage forms, the containermeans, and the written instructions for use. Such packaging means maytake the form of a cardboard or paper box, a plastic or foil pouch, etc.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examples are forthe purpose of illustration only and are not to be construed as limitingthe scope of the invention in any way.

EXAMPLE Pharmacokinetic Enhancement of R7227 In Healthy Adults

Subjects were screened for participation in this study within 21 daysbefore dosing. The study enrolled 14 healthy volunteers (n=14 pergroup). The dosing schedule is illustrated below:

TABLE I Study Day Procedure 1 2 3 4 to 11 12 R7227 dosing¹ x x xRitonavir Dosing² x x x PK collections 24-h PK 48-h PK 48-h PK ¹R7227100 mg single oral dose ²Ritonavir 100 mg dose orally every 12 h.

R7227 formulation—R7227 was formulated into clear size 10 oval softgelatin capsules for oral administration at a strength of 100 mg percapsule (anhydrous free acid equivalent). The capsule fill solutionconsists of R7227-001, polyethylene glycol PEG400 (Macrogol 400), andbutylated hydroxytoluene (BHT). All excipients are compendial grade (NFor EP). Gelatin Type 195 (NF, EP) is used as the bulk gel mass for thecapsule shell, with small amounts of sorbitol liquid 85/70/00 (NF, EP)and water (USP) used as plasticizers.

All study medications were with a meal. On the R7227 administration days(alone or with ritonavir), study medications were administered aftercompletion of a standard high-fat breakfast and the breakfasts on thesedays were identical. R7227 was administered orally as a single dose of a100 mg soft gel capsule on days 1, 3 and 12. On days 3 and 12, R7227 wasadministered with the morning dose of ritonavir. Ritonavir 100 mg wasadministered orally twice daily (every 12 hours) from day 3 to day 12.

Blood samples (5 mL) were collected to determine plasma concentrationsof R7227 (and metabolites when assays were available) according to thefollowing schedule

Day 1, PK samples (5 mL) were be collected before R7227 dosing (predose)and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, and 24 hours afterR7227 dosing;

Days 3 and 12, PK samples (5 mL) were collected before R7227 dosing(predose) and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 24, 36, and48 hours after R7227 dosing.

Primary PK parameters of R7227 that were determined are C. andAUC_(0→∞). Secondary PK parameters of R7227 that were measured includeT_(max) and AUC_(0→last), CL/F, t_(1/2), C_(12 hr) and C_(24 h).

Analysis of variance (ANOVA) was applied to the log-transformed primarypharmacokinetic parameters. Two-sided 90% confidence intervals for theratios of the geometric means of the primary parameters (AUC_(0→∞) andC_(max) of R7227) were derived for the following comparisons:(R7227+Ritonavir) Day 3 versus R7227 Day 1 and (R7227+Ritonavir) Day 12versus R7227 Day 1

Analysis of variance (ANOVA) was used to analyze all primary studyparameters using the following model:

Yij=μ+τi+sj+εij

where Yij denotes the PK parameter to be analyzed, μ denotes the generalmean of the transformed variable, τi, the fixed effect of treatment; sjthe random effect of subject; εij (error). The random deviations εij areassumed to be independent and normally distributed with zero mean andcommon variance σ². The group comparisons τ R7227+Ritonavir—τR7227, theresidual variance σ², and the 90% confidence limits for the groupcomparisons were estimated from the ANOVA model. For log-transformedvariables (AUC_(0→∞) and C_(max)), the ratio of true group means and theconfidence limits for the corresponding ratio of means of theuntransformed variables will be calculated by exponentiation of theleast squares means differences and the confidence limits for thetransformed values, respectively.

TABLE II Geometric Least Square Mean (GLSM) Day 3 Day 12 Ratio of GLSM(90% CI) R7227 Day 1 (with Ritonavir (with Ritonavir PK (unit) N (Alone)single dose) multiple dose) Day 3 vs Day 1 Day 12 vs Day 1 AUC_(0→∞) 12 14.4 82. 79.4 5.70 (4.07, 7.99) 5.50 (4.05, 7.48) ng*h/mL C_(max) 12 8.40 26.4 27.3 3.14 (1.87, 5.25) 3.25 (2.12, 4.96) (ng/mL) C_(12 h) 8^(a) 0.02 1.00 0.53 50.6 (25.6, 100) 26.9 (17.7, 41.0) (ng/mL)C_(12 h) 12^(b) 0.01 0.55 0.36 65.2 (34.8, 122) 42.4 (25.7, 70.0)(ng/mL) ^(a)Non-missing, non-BLQ (0.010 ng/mL) data from all threetreatment days. (BLQ = Below Limit of Quantitation) ^(b)Includeextrapolated C_(12 h) values from 4 subjects with BLQ values on day 1

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

We claim:
 1. A method of inhibiting in vivo oxidative metabolism of aHepatitis C virus NS3/4A protease inhibitor according to formula I in apatient comprising co-administering

a compound of formula I and a cytochrome P450 monooxygenase wherein thecytochrome P450 monooxygenase inhibitor is ritonavir.
 2. The methodaccording to claim 1 wherein the compound of formula I and ritonavir arein separate dosage forms.
 3. The method according to claim 2 wherein theseparate dosage forms are administered about simultaneously.
 4. Themethod according to claim 1 wherein the compound of formula I andritonavir are administered in a single dosage form.
 5. The methodaccording to claim 2 wherein the dose of ritonavir is 50 to 400 mg/dayand the dose of compound I was 25 to 600 mg/day.
 6. A method accordingto claim 6 wherein the dose of ritonavir is 100 to 200 mg/day and thedose of compound I was 50 to 300 mg/day.
 7. A method for treating aHepatitis C virus infection in a patient in need thereof comprisingadministering to the patient in need thereof a therapeutically effectiveamount of a Hepatitis C virus NS3/4A protease inhibitor of formula I, orfree base or other pharmaceutically acceptable salt thereof, and acytochrome P450 monooxygenase inhibitor wherein the cytochrome P450monooxygenase inhibitor is ritonavir.
 8. The method according to claim 7which method comprises co-administering the compound of formula I andritonavir and at least one additional agent selected from animmunomodulatory agent and/or an antiviral agent and/or anotherinhibitor of HCV NS3/4A protease and/or an inhibitor of NS5B polymeraseand/or a broad-spectrum viral inhibitor and/or another cytochrome P-450inhibitor.
 9. The method according to claim 8, wherein saidimmunomodulatory agent is α-, β- or γ-interferon or thymosin, saidantiviral agent is ribavirin or said polymerase inhibitor is R7128. 10.A pharmaceutical composition according to claim 1, and at least onepharmaceutically acceptable excipient, diluent or carrier.
 11. A kitcomprising a Hepatitis C virus NS3 protease inhibitor according toformula I and ritonavir.
 12. A pharmaceutical pack containing comprisinga compound according to formula I and ritonavir together with aninformation insert containing directions for the use of the inhibitors.